PROJECT SUMMARY The directed migration of cells is an important cellular behavior key to normal physiology and deregulated in disease, including cancer. Yet, how cells direct their movements in response to migration cues is not understood. The mechanistic Target of Rapamycin Complex 2 (mTORC2) plays an evolutionarily conserved role in regulating the actin cytoskeleton and controlling the migration of cells. mTORC2 is one of two multiprotein signaling complexes formed by the mTOR kinase. mTORC1 is a key regulator of cell growth and proliferation, and its regulation and signaling pathway are well described. Much less is known about mTORC2, but recent research revealed a role for mTORC2 in promoting cancer cell migration and tumor dissemination. The ability to target the mTORC2 pathway in cancer therapy, however, is greatly hampered by a lack of mechanistic insight into how mTORC2 is activated, regulated, and signals to the cell motility machinery. We aim to address this knowledge gap by identifying the key molecular mechanisms that direct mTORC2 activity and function in cell migration. Recently, we discovered that two Ras family GTPases, Ras and Rap1, bind mTOR and the mTORC2 component RIP3/SIN1, respectively, and control mTORC2 activity in the experimental model Dictyostelium discoideum. Importantly, our preliminary data indicate that these mTORC2 regulatory mechanisms are conserved in human cells. Our overall objective is to determine the mechanism and role of Ras and Rap1-mediated regulation of mTORC2 activity and function in cell migration. Our central hypothesis is that Ras and Rap1 independently regulate mTORC2 in response to a migration signal through distinct interactions with components of mTORC2, thereby controlling mTORC2's signaling functions in cell migration and playing a key role in promoting cancer cell migration. We will test our hypothesis in three specific aims. In Aim 1, we will take advantage of the mTOR interaction with the Dictyostelium Ras protein RasC that we have identified to define the mechanism and role of Ras-mediated mTORC2 regulation in cell migration, using Dictyostelium as experimental model. In Aim 2, we will identify the role of Rap1 in regulating mTORC2 function in cell migration, using prototypical human epithelial cells as experimental model. In Aim 3, we will define the mTORC2 pathway controlling breast cancer cell migration, applying our findings from the mechanistic studies performed in Aim 1 and Aim 2 to specifically interrogate the role of Ras- and Rap1- regulated mTORC2 in promoting the migration of breast cancer cells. Altogether, the proposed work will lead to the description of novel molecular mechanisms involved in regulating mTORC2 and cell migration, including that of cancer cells, which will provide innovative opportunities for the development of therapeutic strategies for inhibiting the migration of cancer cells in metastasis. Furthermore, our findings will also advance the development of treatments for other diseases that involve the pathological migration of cells, including cardiovascular, inflammatory and autoimmune disorders, in which mTOR pathways play important roles.